Mouse adrenal tumor cells (Y-l) in monolayer culture will be used to study questions related to their invasive properties, to the regulation of their steroidogenic activity and to the relationship between these two features of the cells as follows: What is the function of the newly synthesized proteins found associated with mitochondria after addition of ACTH of Y-l cells? What effect do alterations in plasma membrane viscosity and relocation of membrane receptors in an electric field have on the initial events associated with responses of the cells to ACTH and cholera toxin? To what extent can the enhancement of growth and steroidogenesis produced by injection of cells into LAFl mice be duplicated by addition of pituitary extracts to the cells in culture? To assist in answering these questions, the purification of cytochrome P-450 side-chain cleavage will be completed to characterize its properties and to develop a radiommunoassay for this rate-determining enzyme. The relationship between the new protein(s) and transport of cholesterol to mitochondria will be studied by incubating (3H) cholesterol oleate - low density lipoprotein with cells in which side-chain cleavage is blocked and measuring mitochondrial (3H) cholesterol. Membrane receptors will be labeled with fluorescent ligands (ACTH, cholera toxin and concanavalin A) after exposure to an electrical field to estimate membrane viscosity. Cholesterol content of cell membranes will be altered by incubation with lipid-free plasma or cholesterol-containing liposomes. Steroidogenesis and growth of cells in the presence of pituitary extracts will be compared with the response to animal passage. Nonionic detergents will be used to improve extraction of mitochondrial P-450 from Y-l cells. Throughout, comparison will be made between Y-l cells and normal adrenal cells.